Characterization of EiEPSPS Genes from Eleusine indica (L.) Gaertn. Ecotypes Differing in Glyphosate Resistance

Abstract

Goosegrass (Eleusine indica (L.) Gaertn.) is among the most noxious weeds, particularly during the early growth stages of oil palm. Glyphosate is the most widely used herbicide globally for controlling E. indica by targeting the EPSPS enzyme. This study aimed to assess glyphosate resistance of E. indica from several oil palm plantations in West Java using bioassays and molecular studies. Ecotypes exhibiting altered sensitivity were subjected to DNA isolation using the CTAB method to analyze genetic variation in the EiEPSPS gene to determine whether resistance was conferred via a target-site resistance (TSR) mechanism. Bioassays indicated the Cicareuh ecotype (resistance-to-susceptible ratio, R/S 2.6) is classified as decreasing sensitivity, while Mekarjaya (R/S 3.6), Sukasirna (R/S 3.3), Prabugantungan (R/S 3.9), and East Rangkasbitung (R/S 4.7) are classified as low-resistance ecotypes. Molecular analysis revealed region-specific nucleotide variations in both introns and exons. Four nucleotide substitutions were identified in the non-coding regions at positions 1,781, 1,810, 1,843, and 2,844. Within the coding regions, two homozygous non-synonymous mutations were detected, resulting in amino acid substitutions at position 1,375 (Pro-106-Ser) and 3,422 (Pro-381-Leu). Our study indicates that previously reported homozygous nucleotide variations at substitution sites are insufficient to confirm glyphosate resistance, as the same alleles were detected in both resistant and sensitive genotypes. Combined bioassay and molecular analyses suggest that reduced glyphosate sensitivity in E. indica populations sampled from Bogor, Sukabumi, and Lebak has not yet been consistently associated with known EiEPSPS target-site mutations.